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Cotton leaf curl disease is caused by several monopartite begomoviruses and is the major threat to cotton production in the Indian subcontinent. The disease has been shown to be associated with four distinct species, including Cotton leaf curl Kokhran virus (CLCuKoV), and a specific betasatellite-Cotton leaf curl Multan betasatellite (CLCuMuB). Transgenic Nicotiana benthamiana plants were produced which constitutively express the Escherichia coli phage M13 encoded, sequence nonspecific single-stranded (ss) DNA-binding protein, G5 alone and fused with the maize opaque-2 nuclear localization signal (NLS), to evaluate resistance against CLCuKoV-CLCuMuB. Transgenic plants expressing only G5 performed poorly exhibiting symptoms of infection and high virus DNA levels upon inoculation with CLCuKoV and CLCuKoV with CLCuMuB. In contrast, plants transformed with G5 fused to the NLS developed mild symptoms and showed a reduction in virus and betasatellite DNA levels in comparison to nontransformed plants. The results show that G5 may be useful in developing broad-spectrum resistance against ssDNA viruses.